Staphylococcus aureus is a leading cause of drug resistant nosocomial and community acquired infections. The long-term goal of this project is to create an antimicrobial formulation for topical use that is refractory to resistance development by staphylococci. Peptidoglycan hydrolases degrade the major structural component of bacterial cell walls. When applied externally, these enzymes can lyse Gram-positive pathogens with near species-specificity. The goal of this project is to create unique chimeric peptidoglycan hydrolases consisting of three lytic activities, each targeting a unique bond of the staphylococcal cell wall. The hypothesis is that when three unique lytic domains are fused, the chimeric protein would be highly refractory to resistance development in the target pathogen. This hypothesis is based on the observation that: a) no bacterial host has been identified that can resist the lytic action of phage peptidoglycan hydrolase enzymes;b) peptidoglycan hydrolase activity domains are ~200 amino acids in size and can maintain their parental specificities when fused;and c) S. aureus is unlikely to develop three compensatory mutations to resist the action of a triple hydrolase fusion protein. The specific aims are to: 1) Construct multiple stable triple hydrolase fusion proteins, each with three unique staphylococcal cell wall degrading activities. In vitro activity of the chimeric proteins will be optimized. 2) As a means to identify potential resistance mechanisms, investigate the susceptibility of various S. aureus and S. epidermidis strains to the bactericidal activity of the chimeric peptidoglycan hydrolases. Planktonic and biofilm-associated staphylococci will be tested for susceptibility, and in vitro selection for strains resistant to the chimeric peptidoglycan hydrolases will occur. 3) Evaluate chimeric hydrolases for eradication of S. aureus nasal colonization in a rodent model of nasal carriage. Investigate the immune response to the proteins and identify resistant S. aureus mutants that arise in vivo. If resistance to the chimeric PG hydrolases arises in vitro or in vivo, the resistant S. aureus isolates will be analyzed for alterations in PG structure, surface polymer content, and protease production. This investigation may lead to a topical agent to eradicate S. aureus nasal colonization, a documented risk factor for staphylococcal infection. Staphylococci are important bacterial pathogens that cause severe local and systemic infections in humans. The incidence of antibiotic-resistant S. aureus has increased markedly in the hospital and community. This study will explore a novel antimicrobial agent (a triple action chimeric viral enzyme) to eradicate S. aureus nasal colonization, since nasal carriage is a known risk factor for infection.